Methods for detecting cardiac damage

ABSTRACT

The present invention relates to a method for detecting heart damage in a patient. The invention also relates to methods for treatment of patients identified as having heart damage. The invention further pertains to methods for evaluating the efficacy of an ongoing therapeutic regimen designated to treat a damaged heart in a patient.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 15/674,053, filed Aug. 10, 2017, which is a continuation ofU.S. patent application Ser. No. 15/053,284, filed on Feb. 25, 2016 (nowU.S. Pat. No. 9,757,429), which is a continuation of U.S. patentapplication Ser. No. 14/153,695, filed Jan. 13, 2014 (now U.S. Pat. No.9,329,171), which is a continuation of U.S. patent application Ser. No.12/451,397, filed Mar. 26, 2010 (now U.S. Pat. No. 8,628,929), which isa National Stage Application claiming the priority of PCT ApplicationNo. PCT/US2008/006060 filed May 12, 2008, which in turn, claims priorityfrom U.S. Provisional Application No. 60/928,541, filed May 10, 2007.The entire contents of these applications are each incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the field of medical diagnostics. Moreparticularly, the invention is directed to a method for detecting heartdamage in a patient. The invention also relates to methods for treatmentof patients identified as having heart damage. The invention alsopertains to methods for evaluating the efficacy of an ongoingtherapeutic regimen designed to treat a damaged heart in a patient.

BACKGROUND OF THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Heart failure is causally related to a number of conditions that damagethe heart, including coronary heart disease, with or without a heartattack; hypertension; diseases, infections, or toxins that affect theheart muscle, and diseases of the heart valves. The onset of heartfailure can occur rapidly, over days to weeks, but more frequentlydevelops slowly over the course of years, as the heart gradually andprogressively weakens.

Therapeutic intervention directed to reduction of cancer cell load in apatient frequently, if not always, is accompanied by a range ofdeleterious side effects. Indeed, cytostatic agents used aschemotherapeutics for the treatment of various cancers frequentlyexhibit potentially lethal side effects, including cardiotoxicity.Agents commonly used in cytostatic therapy include the anthracyclinesdaunorubicin and prodrugs thereof zorubicin, doxorubicin (adriamycin)and epirubicin, and the synthetic antibiotic mitoxantrone.Anthracyclines, for example, represent a class of chemotherapeuticagents based on daunosamine and tetra-hydro-naphthacene-dione. Thesecompounds are used to treat a variety of cancers, including leukemiasand lymphomas, and solid tumors of the breast, uterus, ovary, and lung.In addition to the expected adverse reactions observed in patientsundergoing chemotherapy, such as hair loss and nausea, therapeuticintervention involving anthracycline administration is complicated andlimited by the marked cardiotoxicity of this class of compounds.Cardiotoxicity associated with anthracycline use is correlated with thetotal dose administered and is frequently irreversible. The cytostaticeffects and cardiotoxicity of these compounds are due, at least in part,to alterations in membrane fluidity and permeability caused byanthracycline binding to components of the cell membrane. Free radicalformation in the heart and accumulation of anthracycline metabolites arealso thought to contribute to heart damage. Cardiotoxicity oftenpresents in electrocardiogram (EKG) abnormalities and arrhythmias or ascardiomyopathy, which may ultimately lead to congestive heart failure.

SUMMARY OF THE INVENTION

The invention is directed to providing novel diagnostic methods forscreening patients to identify those exhibiting signs of heart damage.Patients so identified can then be treated with pharmaceuticalpreparations for the treatment of heart damage as described herein. In aparticular aspect of the invention, diagnostic methods for screeningpatients to identify those exhibiting signs of damage to the heart dueto, for example, cardiotoxicity, hypertension, valvular disorders,myocardial infarction, viral myocarditis, or scleroderma are presented.In a particular aspect, the invention is focused on identifying patientsexhibiting cardiotoxicity resulting from chemotherapeutic intervention.Classification of such patients serves to identify a subgroup ofpatients in need of therapeutic intervention to alleviate short and longterm effects of cardiotoxicity. The subgroup of patients so identifiedcan be treated with pharmaceutical preparations for the treatment ofheart damage that occurs in connection with the use of cardiotoxic dosesof medicaments or chemicals. Under circumstances wherein the heartdamage identified in a patient is due to an ongoing condition, such as,hypertension, valvular disorders, myocardial infarction, viralmyocarditis, or scleroderma, appropriate pharmaceutical preparations canalso be formulated to treat the patient with heart damage.

The present invention also encompasses a method for stratifying patientsaccording to degree or type of heart damage, knowledge of which guides askilled practitioner to choose appropriate therapeutic regimens. Theinvention also includes a method whereby the efficacy of a therapeuticregimen is evaluated.

The novel methods of the invention are based on the discovery thatchanges in intracellular levels of cardiac troponin I (cTnI) and cardiactroponin T (cTnT) in intact cardiac tissue can be used as indicators forthe presence of cardiac damage. More specifically, the present inventorshave discovered that a decrease in intracellular cTnI and cTnT levels inintact cardiac tissue serves as a diagnostic marker to identify patientsat risk for or experiencing cardiac damage. Cardiac tissue can beexcised from a patient and tested in vitro or analyzed in vivo usingmolecular imaging protocols known in the art.

Using either approach, intracellular cTnI and cTnT levels determined forthe patient's cardiac tissue are compared to those of control cardiactissue that expresses wildtype or normal levels of intracellular cTnIand cTnT. Reduced levels of intracellular cTnI and/or cTnT in apatient's cardiac tissue are readily determined by quantitating proteinlevels, which can be achieved using standard methods, and analyzing theresults to determine if a statistically significant decrease inintracellular cTnI and cTnT levels is apparent in the patient's cardiactissue relative to that of the control. Patients showing evidence ofreduced intracellular cTnI and/or cTnT levels are earmarked fortreatment with appropriate compositions chosen to restore, at least inpart, normal heart function as reflected in an increase in intracellularcTnI and cTnT levels or restoration of normal levels of intracellularcTnI and cTnT.

In an embodiment of the present invention, the control or normalintracellular levels of either cTnT or cTnI in cardiac tissue areestablished by determining the intracellular levels of either cTnI orcTnT in cardiac tissue of a patient with normal heart function. Inanother embodiment of the present invention, the control or normalintracellular levels of either cTnI or cTnT in cardiac tissue areestablished by determining the intracellular levels of either cTnT orcTnI in cardiac tissue of a patient prior to onset of treatment capableof causing heart damage.

In an aspect of the present invention, the heart damage is a result ofcardiotoxicity, hypertension, valvular disorders, myocardial infarction,viral myocarditis, or scleroderma. In a further aspect of the invention,the cardiotoxicity is caused by treatment with a chemotherapeutic agentor radiation.

It is also within the scope of the invention to evaluate the efficacy ofa therapeutic regimen designed to at least partially restore normalheart function by measuring intracellular cTnI and cTnT levels incardiac tissue of a treated patient. In accordance with the presentinvention, an increase in intracellular cTnI and cTnT levels in cardiactissue of a treated patient relative to those determined prior totreatment is a positive indicator that the treatment is acting torestore cardiac function.

It is to be understood that intracellular levels of either cTnI or cTnTin cardiac tissue or intracellular levels of both cTnI and cTnT incardiac tissue may be used as indicators of cardiac tissue activityand/or function. This applies to all aspects of the invention, includingmethods directed to evaluating or diagnosing cardiac damage, methodsdirected to stratifying patients with respect to particular therapeuticregimens, and methods directed to evaluating efficacy of a therapeuticregimen.

In accordance with the present invention decreased levels of cTnI and/orcTnT mRNA in cardiac tissue are also indicative of heart damage and maybe used to stratify patient populations. Partial or complete restorationof normal cTnI and/or cTnT mRNA levels is also, therefore, a positiveindicator of therapeutic efficacy as described above with respect toprotein levels.

The present invention pertains to animals, in general, and moreparticularly, to mammals, and even more particularly to humans.Accordingly, the subject is preferably an animal, including but notlimited to animals such as cows, pigs, horses, chickens, cats, dogs,etc., and is preferably a mammal, and most preferably human.Accordingly, the term “subject” or “patient” may be used to refer to ahuman.

The present invention also encompasses a combination therapeutic regimenwherein GGF2 or an epidermal growth factor-like (EGFL) domain encoded bythe neuregulin gene is administered in conjunction with a proteasomeinhibitor to treat cardiac damage. An exemplary proteasome inhibitor foruse in the present invention is Proscript 519, which is a potent andselective proteasome inhibitor. Other proteasome inhibitors of utilityin the present invention include Velcade™ and lactacystin. Additionalproteasome inhibitors are known to those skilled in the art. Indeed,proteasome inhibitors are already used as therapeutic agents for thetreatment of a number of diseases, including some cancers andneurodegenerative diseases.

Also encompassed by the present invention is the use of GGF2 or anepidermal growth factor-like (EGFL) domain encoded by the neuregulingene in the preparation of a medicament for administration to a patientidentified by the present diagnostic methods as exhibiting damage to theheart. The invention further encompasses the use of GGF2 or an epidermalgrowth factor-like (EGFL) domain encoded by the neuregulin gene incombination with a proteasome inhibitor in the preparation of amedicament for administration to a patient identified by the presentdiagnostic methods as exhibiting damage to the heart.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show survival graphs (A), histograms (B-C), and immunoblots(C). For the survival analysis (A), mice were injected with a singledose of doxorubicin [20 mg/kg, intraperitoneally (i.p.)] with or withoutconcomitant injection of NRG1 (0.75 mg/kg, s.c. daily). Fourteen daysurvival was analyzed by the Kaplan-Meier method. With respect to adetermination of serum creatine kinase (CK) levels (B), serum CK levelswere measured in control, Dox-treated and Dox-NRG1 treated mice fourdays after doxorubicin injection. FIG. 1C shows that NRG1 injectionalleviated doxorubicin-induced down-regulation of cTnI, cTnT and cTnCprotein levels in mice. Mice were treated with doxorubicin (20 mg/kg,i.p.) with or without concomitant NRG1 injection (0.75 mg/kg, s.c.daily), Protein levels of cTnI, cTnT and cTnC were measured by Westernblot analysis five days after doxorubicin treatment.

FIGS. 2A-D show immunoblots probed to detect the indicated proteins,FIG. 2A reveals that NRG1 alleviated doxorubicin-induced down-regulationof cTnI and cTnT protein levels in neonatal rat cardiomyocytes (RNCM).RNCM were treated with doxorubicin (1 uM) in the presence or absence ofNRG1 (20 ng/ml or 50 ng/ml). cTnI and cTnT protein levels were measuredby Western blot analysis 48 hours after doxorubicin treatment. FIG. 2Bshows that inhibition of erbB2 abolished the effects of NRG1 on cTnI andcTnT. RNCM were treated with doxorubicin (1 uM) and NRG1 (20 ng/ml) inthe presence or the absence or AG879 (10 uM) and AG1478 (10 uM). Proteinlevels of cTnI and cTnT were analyzed by Western blot analysis. As shownin FIG. 2C, RNCM were treated with doxorubicin and NRG1 in the presenceof LY294002 (10 uM), Akti (5 uM), PD98059 (50 uM) and Rapamycin (10 nM).cTnI and cTnT protein levels were analyzed by Western blot analysis.FIG. 2D shows that RNCM were treated with doxorubicin ordoxorubicin+NRG1 in the presence of cycloheximide (5 ug/ml), Z-VAD (100uM) or MG132 (10 uM). Protein levels of cTnI and cTnT were measured byWestern blot analysis.

FIGS. 3A-3D show immunoblots (A, C, D), and histograms (B). FIG. 3Apresents results wherein RNCM were treated with doxorubicin (1 uM) inthe presence of inhibitors for different caspases (20 uM). The proteinlevels of cTnI and cTnT were measured by Western blot analysis. FIG. 3Bshows the effects of caspase activation in doxorubicin-treated RNCM.Cells were treated with Dox, Dox+NRG1 or Dox+NRG1+LY. Caspase activationwas analyzed by the caspase activation assay. FIG. 3C shows that NRG1decreased doxorubicin-induced cytochrome c release. RNCM were treatedwith Dox or Dox+NRG1. Cytochrome c release was analyzed by cellfractionation and Western blot analysis. FIG. 3D reveals that NRG1decreased doxorubicin-induced ubiquitinylation of cTnI. RNCM weretreated with Dox or Dox+NRG1. Cell lysates were immunioprecipated withcTnI antibody and probed with ubiquitin antibody.

FIGS. 4A-4B show ethidium bromide stained agarose gels (A) andimmunoblots (B). FIG. 4A reveals that NRG-1 inhibiteddoxorubicin-induced down-regulation of mRNA levels of cTnI, cTnT andcardiac specific transcriptional factors. RNCM were treated with Dox orDox+NRG1, mRNA levels of cTnI, cTnT, GATA4, MEF2e and NKX2.5 wereanalyzed by quantitative RT-PCR. FIG. 4B shows that NRG1 inhibiteddoxorubicin-induced dephosphorylation of translational molecules. RNCMwere treated with Dox, Dox+NRG1 or Dox+NRG1+LY. The phosphorylationlevels of mTOR, P70S6K, S6, 4EBP and EIF4G were analyzed by Western blotanalysis.

FIGS. 5A-5C show a survival graph (A), histograms (B), and an immunoblot(C). FIG. 5A shows a survival analysis in doxorubicin-treated mice withcardiac myocyte-specific overexpression of a dominant negative PI3K(dnPI3K). Mice were treated with a single dose of doxorubicin (20 mg/kg,i.p.) with or without concomitant treatment of NRG1 (0.75 mg/kg, s.c.).Fourteen-day survival was analyzed by the Kaplan-Meier method. FIG. 5Bdepicts hemodynamic measurements in doxorubicin-treated dnPI3K mice.Mice were treated with a single dose of doxorubicin (20 mg/kg, i.p.).Hemodynamic measurements were performed six days after the doxorubicintreatment. FIG. 5C shows cTnI protein levels in dnPI3K mice treated withDox or Dox+NRG1.

FIGS. 6A-D show amino acid amid nucleic acid sequences of GGF2.

FIG. 7 shows amino acid and nucleic acid sequences of EGFL1.

FIG. 8 shows amino acid and nucleic acid sequences, of EGFL2.

FIG. 9 shows amino acid and nucleic acid sequences of EGFL3.

FIG. 10 shows amino acid and nucleic acid sequences of EGFL4.

FIG. 11 shows amino acid and nucleic acid sequences of EGFL5.

FIG. 12 shows amino acid and nucleic acid sequences of EGFL6.

DETAILED DESCRIPTION OF THE INVENTION

Typically, when a patient arrives at a hospital complaining of chestpain, the following diagnostic steps are taken to evaluate the conditionof the patient's heart, and determine the severity of any problemsidentified. To begin, the patient is interviewed to compile acomprehensive list of symptoms so that a health care professional canrule out non-heart related problems. Second, an electrocardiogram (EKG)reading is taken, which records the electrical waves made by the heart.The EKG is an essential tool for determining the severity of chest painsassociated with heart conditions and measuring the degree of damage tothe heart. Blood tests are also performed to detect elevated serumlevels of certain factors, such as the troponins and creatine kinase(CK), and the more cardiac specific isoform of creatine kinase (CK-MB),which are indicative of heart damage. The rise in serum levels of CK,CK-MB, and the troponins is due to the release of these moleculesfollowing cardiac muscle cell death and serves, therefore, as a serummarker of necrosis. As a heart muscle cell dies as a result of prolongedischemia, for example, the cell membrane ruptures, releasing thecytosolic contents into the extracellular fluid space, from whence itenters the lymphatic system, and subsequently the bloodstream. Imagingtests, including echocardiogram and perfusion scintigraphy, may also beused in the context of diagnosis.

The most specific markers of cardiac necrosis available are the cardiactroponins. These proteins axe components of the contractile apparatus ofmyocardial cells. Two cardiac troponins, cTnI and cTnT, have beencommercialized and detection of these markers has proven to be areliable and specific assay for detection of minimal levels ofmyocardial damage. The cardiac troponins, like CK-MB, are released fromdead cardiac muscle cells upon rupture of cell membranes, and areeventually detectable in the blood. Necrosis can occur as a result of aprolonged myocardial ischemia, but can also result from myocardial celldamage from other causes such as infection, trauma, or congestive heartfailure.

The present invention differs from those procedures described in theprior art in a variety of aspects. At the outset, it is directed tomeasuring intracellular levels of cTnI and cTnT in intact cardiactissue, rather than serum levels of these markers. Moreover, the presentinventors have discovered that a decrease in intracellular cTnI and cTnTlevels in intact cardiac tissue serves as a diagnostic marker toidentify patients at risk for or experiencing cardiac damage. Thisapproach stands in marked contrast to measurements of serum levels ofthese markers, an increase of which is indicative of heart damage.Moreover, an increase in serum levels of these markers is an acute ortransient marker of heart damage, whereas measurements of intracellularlevels of cTnI and cTnT in intact cardiac tissue serves as a stablemarker reflective of the condition of the heart. In accordance with thepresent invention, identification of patients exhibiting a decrease inintracellular cTnI and cTnT levels in intact cardiac tissue alsoprovides a screening method with which to stratify patients intocategories for subsequent treatment. Patients showing evidence ofreduced intracellular cTnI and cTnT levels are earmarked for treatmentwith appropriate compositions chosen to restore, at least in part,normal heart function as reflected in restoration of such.

An exemplary therapeutic agent for inclusion in such a composition isglial growth factor 2 (GGF2). The amino acid and nucleic acid sequencesof GGF2 are presented in FIGS. 6A-6D. Therapeutic compositions may alsoinclude other exemplary polypeptides, such as epidermal growthfactor-like (EGFL) domains encoded by the neuregulin gene, as shown inFIGS. 7-12, and described in U.S. Pat. No. 5,530,109, which isincorporated herein in its entirety.

In order to more clearly set forth the parameters of the presentinvention, the following definitions are used:

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, reference to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

The compositions containing the molecules or compounds of the inventioncan be administered for diagnostic and/or therapeutic treatments. Indiagnostic applications, compositions are administered to a patient todetermine if the patient has cardiac damage and/or to stratify thepatient with respect to prospective therapeutic regimens. In therapeuticapplications, compositions are administered to a patient diagnosed ashaving cardiac damage in an amount sufficient to treat the patient,thereby at least partially arresting the symptoms of the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective amount or dose.” Amounts effective for thisuse will depend on the severity of the disease and the weight andgeneral state of the patient.

As used herein, the phrase “control sample of cardiac tissue” refers toa sample of cardiac tissue for which intracellular levels of cTnI andcTnT are within normal range. A normal or wildtype range ofintracellular levels of cTnI and cTnT is established based onexperiments such as those presented herein and known in the art whereincardiac tissue of a subject having healthy heart function, as determinedby a skilled practitioner, is used as the standard against whichunknowns are compared. Standards, representative of normal heats, may,for example, be procured from fresh autopsies performed on cadavershaving no evidence of heart disease.

Similarly, the term “control or normal levels” refers to levelsestablished or determined as described herein and understood in the artto be within a range associated with healthy functionality. With respectto the present invention, healthy functionality refers to healthy heartfunction, which can be assessed by a skilled practitioner using standardprocedures such as measuring systolic and diastolic blood pressure,measuring serum levels of indicator proteins such as CK, CK-MB, and thetroponins, performing an EKG, and/or administering a stress test. Askilled practitioner would be aware of that which is generallyconsidered a normal serum level of these proteins.

Various studies have been presented with respect to serum CK levels, forexample, and general guidelines have been established. In one suchstudy, for example, patients with suspected myocardial infarction (MI)who had a serum creatine kinase level of 280 IU/L or more were verylikely to have had an MI; patients with a serum creatine kinase level of80 to 279 IU/L were likely to have had an MI; patients with a creatinekinase level of 40 to 79 IU/L were less likely to have had an MI; andpatients with a creatine kinase level of less than 40 IU/L were muchless likely to have had an MI. With respect to that which is considereda normal serum level of CK or troponins, it is to be understood thatdifferent hospitals have established standards that vary slightly.Moreover, a skilled practitioner would be cognizant of the acceptedstandard in the particular clinical setting (e.g., particular hospital)in which the practitioner is working.

Troponin is also recognized as a sensitive and specific marker forcardiac injury. Indeed, detection of serum troponin I (sTnI) isconsidered to be more accurate than creatine kinase-MB concentrationsfor the diagnosis of MI and provides more useful prognostic information.Detection of sTnI also permits the early identification of thosepatients with acute coronary syndromes who are at an increased risk ofdeath. sTnI Is more sensitive than creatine kinase-MB concentrations fordetection of minor ischemic myocardial injury in patients with smallincreases of total creatine kinase and avoids the high incidence offalse diagnoses associated with the use of creatine kinase-MB as adiagnostic marker in perioperative MI. In one study, for example,patients with moderate elevations of serum troponin I (0.4-2.0 μg/L) hada significantly higher mortality rate and longer length of intensivecare unit and hospital stays when compared with patients without similarelevations. Within the range of moderately elevated troponinconcentrations, higher titers were associated with increasing mortalityrisk, longer hospital and intensive care unit stays, and a higherincidence of myocardial infarction. Treatment of patients exhibitingmaximum serum troponin concentrations equal to or greater than 2 μg/I,with β-blockers and aspirin improved their prognosis.

With respect to normal levels of intracellular cTnI and cTnT, suchdeterminations are established by evaluating normal heart tissue usingstandards methods for determining protein levels such as those taughtherein and known in the art. Decreased levels of intracellular cTnI andcTnT, such as those indicative of an injured or diseased heart, aredetermined as a dec ease in the levels of these proteins relative to anestablished normal level. By way of example, a decrease of at least 50%in the level of cTnI and/or cTnT in heart tissue being tested fordamage, relative to that of healthy heart tissue (normal control),serves as a positive indicator that the heart tissue being tested isdamaged and a patient from whom the damaged tissue was removed wouldbenefit from therapeutic intervention such as that taught herein. In amore particular example, a decrease of at least 75% in the level of cTnTin heart tissue being tested for damage, relative to that of healthyheart tissue (normal control), serves as a positive indicator that theheart tissue being tested is damaged and a patient from whom the damagedtissue was removed would benefit from therapeutic intervention such asthat taught herein.

A skilled practitioner would also be aware of the large body ofscientific literature pertaining to the activity and levels ofintracellular cTnI and cTnT in normal and diseased heart tissue.Examples of references that pertain to intracellular cTnI and cTnT innormal and diseased heart tissue include: Latif et al. (2007, J HeartLung Transplant 26:230-235); Birks et al. (2005, Circulation 112(9Suppl):157-4); Day et al. (2006, Ann NY Acad Sci 1080:437-450); VanBurenet al. (2005, Heart Fail Rev 10:199-209); de Jonge et al. (2005, Int JCardiol 98:465-470); Wen et al. (2008, J Biol Chem April 22. Epub aheadof print); Li et al. (2008, Biochem Biophys Res Commun 369:88-99);Robinson et al. (2007, Circ Res 101:1266-1273); Solzin et al. (2007,Biophys J 93:3917-3931); (Chen et al. (2007, Cardiovasc Toxicol7:114-121); Solaro et al. (2007, Circ Res 101:114-115); Kubo et al.(2007, J Am Coil Cardio 49:2419-2426); Day et al. (2007, J Mol Med85:911-921); Milting et al. (2006, Mol Call Cardiol 41:441-450); theentire contents of each of which is incorporated herein by reference.

In another embodiment of the invention, an increase in intracellularcTnI and cTnT levels, as determined by assaying levels of the proteinsbefore and during (or after) a therapeutic regimen, demonstrates thetherapeutic efficacy of the regimen and provides evidence that theregimen is promoting restoration of normal heart function. Indeed, anincrease in intracellular cTnI and cTnT levels may be used as asurrogate endpoint (i.e, a biomarker intended to substitute for aclinical endpoint) for improved heart function. If, however,intracellular cTnI and cTnT levels remain at a reduced level or arefurther reduced after therapeutic intervention, a skilled practitionerwould reconsider the merit of the regimen with respect to the patientbeing treated and alter or potentially truncate the therapeutic regimen.

As used herein, “preventing” or “prevention” refers to a reduction inrisk of acquiring or developing a disease or disorder (i.e., causing atleast one of the clinical symptoms of the disease not to develop in asubject that may be exposed to a disease-causing agent, or predisposedto the disease in advance of disease onset.

The term “prophylaxis” is related to “prevention”, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

The term “treating” or “treatment” of any disease or disorder refers, inone embodiment, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment.“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, ‘treating’ or ‘treatment’ refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both.

Invasive Procedures for Isolating Tissue

Myocardial biopsy is a common procedure in which heart tissue isobtained from the heart through a catheter, during thoracotomy, orduring open chest surgery. It is commonly used to diagnose the etiologyof heart failure. The heart tissue is analyzed both histologically andbiochemically. The results of these tests are useful in diagnosing thecause of the heart failure. For review of myocardial biopsy in theclinical setting see Veinot (2002, Can J Cardiol. 18(3):287-96), theentire disclosure of which is incorporated herein by reference.

The results of the myocardial biopsy may indicate that hear, failure isthe result of such causes as scleroderma, viral myocarditis, drugtoxicity or any number of causes of heart failure. This diagnosis willdictate what, if any, therapeutic intervention may be useful and shouldbe employed.

Myocardial biopsy (or cardiac biopsy) is an invasive procedure, whereinat bioptome (a small catheter with a grasping device on the end), forexample, may be used to obtain a small piece of heart muscle tissue thatcan be analyzed. Myocardial biopsy may be used to evaluate hearttransplant rejection and to diagnose myocarditis (inflammation of theheart).

Although a skilled practitioner would be aware of the basic protocol formyocardial biopsy using a catheter, it essentially involves thefollowing: a local anesthetic is used to numb part of the neck or groinof a patient; a practitioner inserts a plastic introducer sheath (ashort, hollow tube through which the catheter is placed) into a bloodvessel in the numbed region; a bioptome is inserted through the sheathand threaded to the right ventricle of the patient; and samples arecollected from the heart using the grasping device of the bioptome.During the procedure, an x-ray camera is generally used to position thebioptome properly. Samples are about the size of the head of a pin. Whena sufficient number of samples have been collected, the catheter isremoved and localized bleeding is controlled by firm pressure. Patientsmay be awake and conscious during the procedure.

Certain proteins have routinely been measured in the blood streamfollowing a myocardial event to predict and diagnose if and the extentto which the myocardium has been damaged. These proteins include, butare not limited to, creatine kinase and troponin. Detecting specificsub-types of these and other proteins in the blood stream is diagnosticof release of the proteins from the myocardium and thus damage.Detection of these indicator proteins in the sera is used routinely inthe acute setting where a cardiac event is suspected and have provenuseful in determining the best treatment for the patient. While usefulin the acute setting, cardiac protein levels in the blood stream have novalue in diagnosing or predicting heart failure more than a few daysafter a cardiac event, nor do they have any value in determining theetiology of heart disease or proper treatment course. As cardiac proteinlevels are not elevated in the blood except for immediately following acardiac event, measurement of these proteins in the blood stream revealslittle regarding the state of the myocardium.

In contrast, the present invention describes the use of myocardialbiopsy to predict which patients may be responsive to acardioprotective, cardiorestorative and other heart failure therapy.Predicting which patient will respond to these therapies will improvetreatment by helping to ensure that the correct patients receivespecific therapies and, significantly, limit the number of patients thatreceive therapies that will have little value and thus only expose themto the risk of potentially serious side effects. Additionally,myocardial biopsy and determination of certain protein content maydemonstrate which patients are responding to certain therapies and withwhom treatment should be continued.

The data presented herein show that, as predicted, immediately followingcardiac challenge certain proteins, including creatine kinase andtroponin can be detected in the blood stream. The data also demonstratethat one can measure a stable decrease in myocardial proteins fromsamples of myocardium taken long after the cardiac proteins in the bloodstream have returned to nor-mal low levels. These data also show thattreatment of failing hearts with neuregulin can prevent the decline andrestore the cardiac protein content of the diseased heart. Thisrestoration correlates with improved survival and cardiac physiology.

Moreover, these data demonstrate that neuregulin can restore thetroponin content of the myocardium following insult. Troponin is a keyprotein essential for the contractile properties of the heart.Restoration of the troponin content is thus important for restoration ofnormal cardiac physiology. The use of myocardial troponin contentmeasurement in a myocardial biopsy to determine if reduced troponinmyocardial content is a component of the reduced cardiac function willhelp predict which patients may respond to a cardiorestorative therapysuch as administration of a neuregulin. Additionally, myocardialtroponin content measurement may be used as an assay for determiningwhich patients are responding to a cardiorestorative therapy such as aneuregulin. Knowing when patients are responding favorably to thetherapy will help predict if and when such patients should be treatedagain, observed or removed from therapy. Myocardial troponin contentmeasurement may be used to optimize dosing for individual patients thatare being treated for heart disease with a cardiorestorative treatment.Similarly, in a chronic disease state in which continuous decline ofcardiac troponin levels occur, maintenance of myocardial troponin levelsmay indicate success of a cardioprotective therapy such as a neuregulinin the presence of ongoing disease. In this manner myocardial troponinlevels may similarly be used to predict response to therapy andoptimization of dosing.

Dosing for GGF2 or EGFL domains of neuregulin, for example, can beinitiated at about 100 mg/kg and dosing thereof titrated to higherlevels based on patient response, including an assessment ofintracellular levels of cTnI and cTnT in cardiac tissue of the patient.

Once isolated, intracellular levels of cTnI and cTnT in cardiac tissuecan be determined by methods described in the Examples presented hereinand known in the art. Cellular lysates of isolated cardiac tissue orprecipitates derived therefrom may, for example, be analyzed using avariety of techniques including, but not limited to, immunoblotanalysis, enzyme-linked immunosorbent assay (ELISA), and massspectrometry. Indeed, an ELISA protocol has been developed to detectcardiac-specific troponin T that utilizes a high-affinity,cardiac-specific antibody (M11.7). The detection limit of this assay islower than that of first generation ELISA protocols that used thecross-reacting antibody 1B10 (0.0123 μg/L versus 0.04 μg/L,respectively).

Methods for Detecting Troponin I and T Levels In Vivo

Technological advances in the field of molecular imaging have madepossible noninvasive, high-resolution in vivo imaging techniques thatenable clinicians to diagnose and evaluate therapeutic efficacy on amolecular and cellular level. The term molecular imaging is, indeed,“broadly defined as the in vive characterization and measurement ofbiological processes at the cellular and molecular level”. SeeWeissleder et al. (Radiology 219:316-333, 2001), which is incorporatedherein in its entirety. Nuclear imaging, for example, which includespositron emission tomography (PET), micro-PET, single photon emissioncomputed tomographic (SPECT), and planar imaging, generally involvesvisualizing an endogenous or expressed protein using specificradiopharmaceuticals as detection probes. PET, for example, is capableof producing a three-dimensional image or map of functional processes inthe body which facilitates real time analyses of cellular components andmolecular interactions within cells. PET is used as both a medical and aresearch tool. With respect to medical applications, it is usedextensively in clinical oncology to image tumors and detect metastasesand in clinical diagnosis of a variety of brain diseases, especiallythose associated with dementia. The ability to perform repeated PETanalyses on a patient enables a skilled practitioner to compare resultsover time so as to evaluate, for example, disease progression orefficacy of a selected treatment protocol. PET has also been used as aresearch tool to map normal human brain and hart function.

Alternative methods of scanning include x-ray computed tomography (CT),magnetic resonance imaging (MRI), functional magnetic resonance imaging(fMRI), and ultrasound. Imaging scans such as CT and MRI are well suitedto visualize organic anatomic changes in the body, whereas, as indicatedabove, PET scanners, like SPECT and MRI, have the resolution to detectchanges on a molecular level, even in advance of changes evident on theanatomic level. Imaging scans such as PET achieve this end by usingradiolabeled molecular probes that exhibit different rates of uptake,depending on the type and function of the tissue of interest.Alterations in regional blood flow in various anatomic structures canalso be visualized and quantified with a PET scan, Some of the abovescanning techniques can be used in combination, depending oncompatibility of radioisotopes utilized, so as to provide morecomprehensive information that improves the accuracy of the clinicalassessment. This is discussed herein below with respect to SPECTimaging.

As indicated above, nuclear imaging techniques such as PET, micro-PET,SPECT, and planar imaging, are directed to visualizing an endogenous orexpressed protein using specific radiopharmaceuticals as detectionprobes. Imaging marker genes that encode intracellular enzymes andimaging marker genes that encode cell surface proteins or receptors havebeen used successfully in a variety of experimental systems to imagespecific molecules in vivo. The activity of an intracellular enzyme mayalso be assessed by labeling by-products indicative of the level and/oractivity of a particular intracellular enzyme. PET and microPET, forexample, use positron-labeled molecules to image processes involved inmetabolism, cellular communication, and gene expression. Molecularimaging technologies are described in Kim (Korean J Radiology 4:201-210,2003), which is incorporated herein by reference in its entirety.

Radionuclides used in PET scanning are typically isotopes with shorthalf lives such as ¹¹C (˜20 min), ¹³N (˜10 min), ¹⁵O (˜2 min), and ¹⁸F(˜110 min). These radionuclides are incorporated into compounds normallyused by the body such as glucose, water or ammonia and then injectedinto the body to trace where they become distributed. Such labeledcompounds are known as radiotracers. The short half-life of theseradiotracers restricts clinical PET primarily to the use of tracerslabeled with ¹⁸F, which has a half life of 110 minutes and can betransported a reasonable distance before use, or to ⁸²Rb, which can becreated in a portable generator and is used for myocardial perfusionstudies.

SPECT is an example of a nuclear medicine tomographic imaging techniquethat uses gamma rays. It is very similar to conventional nuclearmedicine planar imaging using a gamma camera. Unlike planar imaging,however, SPECT can provide true 3D information. This information istypically presented as cross-sectional slices through the patient, butcan be modified with regard to presentation as required. The imageobtained by a gamma camera image is a 2-dimensional view of3-dimensional distribution of a radionuclide, Because SPECT acquisitionis very similar to planar gamma camera imaging, the sameradiopharmaceuticals may be used for either protocol. If a patient isexamined using an alternative type of nuclear medicine scan, forexample, but the images are non-diagnostic, it may be possible toperform a subsequent imaging scan using SPECT by reconfiguring thecamera for SPECT image acquisition while the patient remains on thetable or moving the patient to a SPECT instrument.

SPECT can also be used for cardiac gated acquisitions. To obtaindifferential information about the heart in various parts of its cycle,gated myocardial SPECT can be used to obtain quantitative informationabout myocardial perfusion, thickness, and contractility of themyocardium during various parts of the cardiac cycle. It is also used tofacilitate calculation of left ventricular ejection fraction, strokevolume, and cardiac output.

Myocardial perfusion imaging (MPI) is an example of functional cardiacimaging, which is used for the diagnosis of ischemic heart disease. Itis based on the principle that impaired or diseased myocardium receivesless blood flow than normal myocardium under conditions of stress inbrief, a cardiac specific radiopharmaceutical is administered, forexample, ^(99m)Tc-tetrofosmin (Myoview™, GE healthcare) or^(99m)Tc-sestamibi (Cardiolite®, Bristol-Myers Squibb), after whichadministration the heart rate is raised to induce myocardial stress. Inaccordance with standard practice, enhanced heart rate is typicallyeither exercise induced or pharmacologically induced with adenosine,dobutamine or dipyridamole. SPECT imaging performed after induction ofstress reveals the distribution of the radiopharmaceutical, andtherefore the relative blood flow to the different regions of themyocardium. Diagnosis is made by comparing stress images to a subsequentset of images obtained at rest.

In that troponin is a structural component of the cytoskeleton and anenzyme, molecular imaging techniques are envisioned to measureintracellular cTnI and cTnT levels in vive.

Indeed, cardiac troponin T is present in myocytes at highconcentrations, both in cytosolic and structurally-bound protein pools.The cytosolic pool amounts to 6%, whereas the amount in myofibrilscorresponds to 94% of the total troponin T mass in the cardiomyocyte. Inview of the high levels normally present in myocytes, labeling of eitheror both pools of troponin T will generate sufficient signal to bevisualized with a reasonable degree of accuracy and resolution.

One potential approach envisioned for visualizing troponin complexes incardiac tissue in vive takes advantage of the calcium (Ca²⁺) bindingproperties of this complex. It is known that contraction is initiated byCa²⁺ binding to troponin (Tn) in striated muscle, and more particularlyby the Ca²⁺-binding subunit of Tn (TnC), which leads to myosin bindingto actin, force generation and shortening. The level of force isregulated by the availability of myosin binding sites on the thinfilament, which is controlled by the position of tropomyosin (Tm) on thesurface of actin. The inhibitory subunit of Tn (TnI) binds to actin inthe absence of Ca²⁺, anchoring Tm so as to inhibit myosin binding. WhenCa²⁺ binds to TnC, it induces strong TnI-TnC interactions and weakensthe TnI-actin interactions, resulting in increased mobility of Tm andexposure of strong myosin binding sites on actin. In addition, myosinbinding to actin leads to crossbridge formation, which is thought tofurther displace Tm from blocking positions and is necessary for maximalactivation of thin filaments in skeletal muscle.

In view of the above properties, a skilled practitioner would envisionutilizing a Ca²⁺ radionuclide to visualize cytoskeletal components and,more particularly, troponin levels in cardiac tissue in vivo.Alternatively, small molecules that specifically recognize either cTnIor cTnT may be labeled with radionuclides and administered to patientsto visualize intracellular levels of these proteins. A skilledpractitioner could envision a variety of labeled probes of utility inthe present method, including ligands, antibodies, and substrates ofcTnI or cTnT and/or proteins that interact with cTnI or cTnT.

Radiopharmaceuticals such as those described in U.S. Pat. No. 5,324,502may also be used to advantage in the present method for imagingmyocardial tissues. As described in U.S. Pat. No. 5,324,502, suchradiopharmaceuticals are prepared by firming lipophilic, cationiccomplexes of radioactive metal ions with metal chelating ligandscomprising the Schiff base adducts of triamines and tetraamines withoptionally substituted salicylaldehydes. These lipophilic, cationic,radioactive complexes exhibit high uptake and retention in myocardialtissues. Preferred gallium-68(III) complexes in accordance with thisinvention can be used to image the heart using positron emissiontomography. In an aspect of the present invention, suchradiopharmaceuticals may be used as a means for targeting uptake ofagents that bind to the troponins to cardiac tissue.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an agent,and a pharmaceutically acceptable carrier. In a particular embodiment,the term “pharmaceutically acceptable” means approved by a regulatoryagency of the federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin, incorporated in its entirelyby reference herein. Such compositions contain a therapeuticallyeffective amount of the compound, preferably in purified form, togetherwith a suitable amount of carrier so as to provide a form for properadministration to a subject. The formulation should suit the mode ofadministration.

In a particular embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings, Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment of a heart damage can be determined by standard clinicaltechniques based on the present description. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each subject's circumstances. However, suitabledosage ranges for intravenous administration are generally about 20-500micrograms of active compound per kilogram body weight. Suitable dosageranges for intranasal administration are generally about 0.01 μg/kg bodyweight to 1 mg/kg body weight. Suppositories generally contain activeingredient in the range of 0.5% to 10% by weight; oral formulationspreferably contain 10% to 95% active ingredient. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu (1987)J. Biol. Chem. 262:4429-4432), and construction of a nucleic acid aspart of a retroviral or other vector. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally, e.g., by localinfusion during surgery, topical application, e.g., by injection, bymeans of a catheter, or by means of an implant, said implant being of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

In another embodiment, the compound or agent can be delivered in avesicle, in particular a liposome (see Langer (1990) Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or agent can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al. (1980) Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol.Chem. 23:61; see also Levy et al, (1985) Science 228:190; During et al.(1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., damaged heart, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

Example I

The present inventors have previously reported that GGF2, a recombinantneuregulin-1, improves survival and cardiac function indoxorubicin-treated mice. As described herein, the present inventorshave investigated whether GGF2 prevents doxorubicin-induced cardiacmyofibril loss in vive and in cardiomyocytes in vitro. The resultspresented herein have led to the novel discovery that the intracellularexpression levels of particular cardiac proteins are useful indicatorsof normal heart function. More specifically, the present inventors havediscovered that changes in the intracellular levels of cardiac troponinI (cTnI) and cardiac troponin T (cTnT) in intact cardiac tissue can beused as indicators of cardiac damage. In a particular aspect of theinvention, a decrease in intracellular cTnI and cTnT levels in intactcardiac tissue has been shown to be a useful diagnostic marker toidentify patients at risk for or experiencing cardiac damage. Suchpatients are then selected for appropriate preventative or therapeuticintervention as described herein. Determination of intracellular cTnIand cTnT levels in intact cardiac tissue may also be used to advantageto evaluate the efficacy of ongoing therapeutic intervention, sincerestoration of normal intracellular cTnI and cTnT levels would serve asa positive indicator that the therapy was improving heart function orrestorative of normal heart function.

Materials and Methods

Materials

C57BL/6 mice and Wistar rats were obtained from Charles RiverLaboratories. Doxorubicin was obtained from Bedford laboratories. Glialgrowth factor 2 was a gift from Acorda Therapeutics, Inc. MG132,cycloheximide and actinomycin were obtained from Sigma. LY294002 and PD98059 were from Cell Signalling Technology. Antibodies were ordered fromthe following vendors: Troponin 1, (GATA4 and Nkx2.5 were from SantaCruz Biotechnology; α-sarcomeric actin, troponin T, troponin C,tropomyosin and cardiac troponin T were from Abeam; Desmin and α-actininwere from Sigma and cardiac troponin I were from GeneTex. MEM, Hank'ssolution and fetal bovine serum were obtained from Invitrogen. All otherreagents for cell culture were obtained from Sigma.

Animal Models

Eight to ten week old C57BL/6 male mice were used for analyses whereinheart samples were isolated. A subacute doxorubicin cardiotoxicity modelwas used for this study. Mice were treated with a single dose ofdoxorubicin (20 mg/kg, i.p.). Twenty-four hours before, on the day, andevery day after doxorubicin treatment, mice were treated with eitherGGF2 (0.75 mg/kg, s.c.) or placebo (formulation buffer for GGF2). Micewere sacrificed 4.5 days after doxorubicin treatment. Heart samples(n=3-4 per group) were collected and snap-frozen in liquid nitrogen.

With respect to those analyses wherein cardiac function was measured (asdetailed below), three-month old C57BL/6 mice were treated with a singledose of doxorubicin (20 mg/kg, i.p.). Glial growth factor 2 (GGF2-Dox,0.75 mg/kg, s.c., n=74) or placebo (the buffer used to dissolve GGF2,placebo-Dox, n=73) was injected into mice one day before and once dailyfollowing doxorubicin treatment. Mice without doxorubicin treatment wereused as controls (n=20). Cardiac function was assessed by direct leftventricular (LV) catheterization four days and two weeks afterdoxorubicin treatment. Two-week survival was analyzed by theKaplan-Meier methods.

Neonatal Cardiac Myocyte Culture

Neonatal cardiac myocytes were dissociated as described previously(Okoshi et al. Journal of Cardiac Failure. 2004; 10:511-518). In brief,ventricles from day 0-day 3 Wistar rats were dissociated in trypsin andDnase II. Cells were washed and pre-plated in 100 mm dishes in MEMcontaining 0.5% fetal bovine serum. After 30 minutes, the myocytes weresuspended in the same medium containing 0.1 mmol/L bromodeoxyuridine andthen plated at the density of 500-1000 cells/mm² in 100 mm culturedishes. Forty-eight hours after dissociation, the medium was changed toserum-free MEM containing 0.1% BSA and cultured overnight beforestimulation.

NRG1 Improved Survival and Cardiac Function in Doxorubicin-Treated Mice.

The present results reveal that two-weeks after doxorubicin treatment,survival in doxorubicin treated mice was significantly decreasedcompared with non-treated control mice. Concomitant treatment with NRG1(Dox-NRG1), however, significantly improved survival indoxorubicin-injured mice compared to placebo-treated mice (Dox-Placebo)(FIG. 1A). Cardiac function was further assessed five days afterdoxorubicin injection, at which point, survival started to decline indoxorubicin-treated mice. As shown in Table 1, the body weight (BW),heart weight (HW) and left ventricular weight (LVW) were significantlydecreased in both Dox-Placebo and Dox-NRG1 mice compared with control(untreated) mice. HW and LVW normalized by tibia length (HW/TL andLVW/TL) were significantly decreased in Dox-Placebo mice compared tocontrol mice. These indices were not, however, different betweenDox-NRG1 and control mice, LV systolic pressure (LVSP), cardiac outputand dP/dt min were significantly decreased in Dox-Placebo mice comparedwith controls. In contrast, these indicators of heart function were notsignificantly different between Dox-NRG1 mice and control mice,indicating an improvement of cardiac systolic function in NRG1(Dox-NRG1) treated mice compared with Placebo treated mice(Dox-Placebo). Serum creatine kinase (CK) levels, an index of cardiacinjury, were also assessed to provide an additional read-out of heartfunction. As shown in FIG. 1B, the CK level was significantly increasedin both Dox-Placebo and Dox-NRG1 mice compared to control mice. The CKlevel was significantly lower in Dox-NRG1 mice, however, as comparedwith Dox-Placebo mice. These results demonstrated that NRG-1 treatmentsignificantly improved survival and cardiac systolic function indoxorubicin-injured mice.

TABLE 1 Hemodynamic measurements in Dox + Placebo and Dox + NRG1 treatedC57BL/6 mice. Control Dox + Placebo Dox + NRG1 Group (n = 8) (n = 9) (n= 8) BW (g)  25 ± 0.4 20 ± 0.4^(†)   21 ± 0.9^(†) TL (mm) 16.7 ± 0.6 16.3 ± 0.2   16.0 ± 0.0  HW (mg) 110 ± 3  83 ± 3^(†)  89 ± 3^(† ) LVW(mg) 88 ± 2  70 ± 1^(†)  75 ± 3^(† ) HW/BW (mg/g) 4.5 ± 0.1 4.1 ±0.1^(†) 4.2 ± 0.1 LVW/BW (mg/g) 3.6 ± 0.1 3.5 ± 0.1  3.6 ± 0.1 HW/TL(mg/mm) 6.6 ± 0.3 5.1 ± 0.1^(†) 5.7 ± 0.2 LVW/TL (mg/mm) 5.4 ± 0.2 4.4 ±0.1^(†) 4.8 ± 0.2 LVSP (mmHg) 100 ± 3  84 ± 2*  91 ± 4  LVEDP (mmHg) 3.9± 1.4 3.6 ± 0.9  4.1 ± 1.1 dP/dt max 10914 ± 856  7067 ± 884   9709 ±1181 (mmHg/sec) dP/dt min 7859 ± 510  5021 ± 602*  7211 ± 725 (mmHg/sec)EF (%) 27 ± 2  17 ± 2^(†)  20 ± 2* Cardiac Output 3276 ± 469  1185 ±203^(†)  2052 ± 423  (ul/min) HR (beat/min) 512 ± 40  407 ± 39   540 ±45  *p < 0.05; ^(†)p < 0.01 vs. Control.NRG1 Alleviated Doxorubicin-Induced Down-Regulation of cTnI and cTnt inthe Heart In Vivo.

One of the mechanisms of doxorubicin-induced cardiotoxicity is loss ofcardiac myofibrils. As described herein, the present inventorsinvestigated whether NRG1 injection in vivo inhibiteddoxorubicin-induced myofibril loss. Results presented herein demonstratethat the levels of cardiac structural proteins, α-sarcomeric actin,α-actinin, troponin T (TnT), troponin I (TnI), troponin C (TnC) andtropomyosin were significantly decreased in doxorubicin-treated hearts.NRG1 injection in vivo significantly increased the protein levels ofcTnI, cTnT and cTnC in doxorubicin-injured hearts (FIG. 1C), but had noeffects on the protein levels of α-sarcomeric actin, α-actinin andtropomyosin.

NRG1 Abolished Doxorubicin-Induced Down-Regulation of cTnI and cTnTProteins in Cardiomyocytes In Vitro.

To further study the mechanism of how NRG1 inhibited doxorubicin-induceddown-regulation of cTnI and cTnT, the present inventors conducted invitro studies using neonatal rat cardiomyocyte culture (NRCM). As shownin FIG. 2A, doxorubicin significantly reduced the protein levels of cTnIand cTnT in NRCM; the presence of NRG-1, however, maintained the levelsof these proteins in doxorubicin-treated cardiomyocytes. These resultsfurther demonstrated that these effects of NRG1 were blocked byinhibitors for erbB2, PI3K, Akt, mTOR or ERK (FIGS. 2B and 2C), but werenot blocked by inhibitors for erbB4 (FIG. 2B), p38 or PKC. The instantresults also showed that the preventative and/or restorative effects ofNRG1 were blocked by cycloheximide, a protein translation inhibitor(FIG. 2D), but not by actinomycin D, a transcription inhibitor.Doxorubicin-induced down-regulation of cTnI and cTnT was abolished byZ-VAD, a pan-caspase inhibitor, and MG132, a proteasome inhibitor (FIG.2D), but not by bafilomycin A1, a lysosome inhibitor.

NRG1 Inhibited Doxorubicin-Induced Caspase and Proteosome Degradation ofcTnI and cTnT in Cardiomyocytes In Vitro.

The maintenance of the level of a protein in the cell is a dynamicprocess. It depends on the rate of synthesis and degradation of aprotein. The present inventors explored the mechanism wherebydoxorubicin decreased the protein levels of cTnI and cTnT to determineif this effect is caused by increasing their degradation and/or bydecreasing their synthesis and whether NRG1 blocked any of these effectsof doxorubicin.

To investigate whether NRG1 inhibited doxorubicin-induced caspasedegradation of cTnI and cTnT, the present inventors first identified thespecific caspases that were responsible for doxorubicin-induceddegradation of cTnI and cTnT. Cardiomyocytes were treated withdoxorubicin in the presence of a specific caspase inhibitor. As shown inFIG. 3A, inhibitors for caspase-3, caspase-6 or caspase-9 (intrinsicpathway) blocked doxorubicin-induced down-regulation of both cTnI andcTnT. In addition, down-regulation of cTnI was blocked by inhibitors forcaspase-10 (extrinsic pathway), caspase-2, caspase-13 or caspase-5. Onthe other hand, the down-regulation of cTnT was also blocked bycaspase-2 and caspase-13. The present inventors then tested whetherdoxorubicin activated and whether NRG1 inhibited the activation of thesecaspases. An in vitro caspase activation assay revealed that doxorubicinsignificantly increased the activation of caspase 3, 6 and 9 as well ascaspase-10, 2, and 5. NRG1 treatment of cardiomyocytes significantlyinhibited doxorubicin-induced activation of these caspases (FIG. 3B).PI3K inhibitor LY294002 abolished these effects of NRG1. The presentinventors further demonstrated that doxorubicin induced increasing ofcytochrome c release to the cytosol in NRCM. NRG1 treatment, however,inhibited this effect of doxorubicin (FIG. 3C). This result, incombination with the findings of caspase-3, 6 and 9 activations,suggested that doxorubicin increased mitochondrial outer membranepermeabilization, which may be responsible for the activation ofcaspase-3, 6 and 9, and NRG1 blocked these effects of doxorubicin. Theseresults demonstrated that NRG-1 inhibited doxorubicin-induced activationof both intrinsic and extrinsic caspase activation, which wereresponsible, at least in part, for the degradation of cTnI and cTnT.

The present inventors further demonstrated that doxorubicin-induceddown-regulation of cTnI was blocked by MG132 (FIG. 2D). In short, thepresent inventors asked whether doxorubicin increased proteasomedegradation of cTnI and whether NRG1 blocked this effect. As shown inFIG. 3D, doxorubicin increased the ubiquitinylation of cTnI; NRG1treatment abolished this effect of doxorubicin. This result demonstratedthat NRG1 decreased doxorubicin-induced proteasome degradation of cTnI.

NRG1 Alleviated Doxorubicin-Induced Decrease in Synthesis of cTnI andcTnT in Cardiomyocytes In Vitro.

To further test whether doxorubicin decreased the transcription of cTnIand cTnT and whether NRG1 reversed this effect of doxorubicin, the mRNAlevels of these proteins in Dox-Placebo and Dox-NRG1 treatedcardiomyocytes were measured. As shown in FIG. 4A, doxorubicin decreasedthe mRNA of both cTnI and cTnT. On the other hand, NRG1 maintained themRNA level of cTnI and cTnT in doxorubicin-treated cardiomyocytes. Inaddition, NRG1 maintained the mRNA level of GATA4 and slightly increasedthe mRNA level of MEF2c and Nkx2.5 (FIG. 4A), which are transcriptionalfactors important for cardiac specific gene transcription.

The present results showed that NRG-1's effects on cTnI and cTnT wereblocked by cyclohexamide (FIG. 2D), suggesting that NRG1 increased thetranslation of these proteins. We assessed the activation of severaltranslation machineries and related signaling pathways in Dox-Placeboand Dox-NRG1 treated cardiomyocytes. As shown in FIG. 4B, 48 hours afterthe treatment, the phosphorylation levels of mTOR (Set 2448). P70S6K(Thr421/Ser424), S6(Ser240/244) and eIF4G (Ser1108) were decreased inDox-Placebo, but were maintained in Dox-NRG1 treated cardiomyocytes.LY294002 blocked these effects of NRG-1. These results suggest that NRG,via PI3K, maintained the activation of protein translational machineriesin doxorubicin-treated cardiomyocytes, which may be responsible formaintaining the protein levels of cTnI and cTnT.

These results demonstrated that NRG1 alleviated doxorubicin-induceddown-regulation of cTnI and cTnT via multiple mechanisms, which includeinhibition of the activation of intrinsic and extrinsic caspases, aswell as inflammatory activated caspases, inhibition of theubiquitinylation of cTnI, and an increase in transcription and theactivation of translation signaling and machineries. These results alsosuggested that PI3K played a major role for NRG1 in maintaining cTnI andcTnT levels in cardiomyocytes.

To further investigate the role of PI3K in mediating NRG1's cardiacprotective effects in vivo, the present inventors used transgenic micewith cardiac myocyte-specific overexpression of a dominant negative PI3Kand treated them with doxorubicin as described above. As shown in FIG.5A, the survival rate was decreased in dnPI3K-Dox-Placebo mice comparedwith WT-Dox-Placebo mice. NRG1 (WT-Dox-NRG) treatment improved survivalin doxorubicin injured WT (WT-Dox-Placebo) mice (67% vs. 33%).Intriguingly, the magnitude of this improvement was greater than thatobserved in C57BL/6 male mice (FIG. 1A). The present results furthershowed that this improvement of the survival rate was dampened indoxorubicin-treated dnPI3K mice (dnPI3K-Dox-NRG: 56% vs. WT-Dox-NRG:67%).

Cardiac function was also evaluated in these mice. As shown in FIG. 5B,LVSP, dP/dt max and dP/dt main, as well as cardiac output were moreseverely depressed in dnPI3K-Dox-Placebo mice compared with dnPI3Kcontrol than WT-Dox-Placebo mice compared with WT mice, indicating moresevere cardiac dysfunction in dnPI3K-Dox-Placebo mice.

The present inventors measured cTnI and cTnT protein levels indoxorubicin-treated WT and dnPI3K mice. Without doxorubicin treatment,the protein levels of cTnI and cTnT were similar in WT and dnPI3Khearts. Two-weeks after the doxorubicin injection, a decrease in cTntprotein levels was observed in dnPI3K-Dox-Placebo, treated heartscompared with non-treated dnPI3K hearts. Surprisingly, NRG1 treatment isstill abolished doxorubicin-induced down-regulation of cTnI in dnPI3Khearts (dnPI3K-Dox-NRG, FIG. 5C). No changes in cTnT protein levels wereobserved in the hearts of doxorubicin-treated mice compared to controlmice at this point.

These results demonstrate that GGF2 specifically maintains TnT and TnIprotein levels in doxorubicin-injured hearts. Moreover, these findingsreveal that GGF2 increases survival of doxorubicin-treated mice and thisis associated with an improvement in cardiac function as evident in micetreated with both doxorubicin and GGF2.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

The invention claimed is:
 1. A method for treating a patient exhibitingheart damage, the method comprising: administering to the patient atherapeutically effective amount of glial growth factor 2 (GGF2) and aproteasome inhibitor, wherein the patient of heart damage is identifiedby a decrease of at least 50% in intracellular levels of either cardiactroponin T (cTnT) or cardiac troponin I (cTnI) protein in cardiac tissueof the patient relative to control or normal intracellular levels ofeither cTnT or cTnI protein.
 2. The method of claim 1, wherein thepatient is a mammal.
 3. The method of claim 2, wherein the mammal is ahuman.
 4. The method of claim 1, wherein the heart damage is a result ofcardiotoxicity, hypertension, valvular disorders, myocardial infarction,viral myocarditis, or scleroderma.
 5. The method of claim 4, wherein thecardiotoxicity is caused by treatment with a chemotherapeutic agent orother therapeutic agent, radiation, or agents used for non-therapeutic,recreational purposes.
 6. The method of claim 1, wherein detectingintracellular level of cTnT or cTnI in the cardiac tissue is performedin vitro.
 7. The method of claim 1, wherein detecting intracellularlevel of cTnT or cTnI in the cardiac tissue is performed in vivo.